DNA and Chromosomes Flashcards
1
Q
What are the structural units (building blocks) of Nucleic acids?
A
Nucleotides
2
Q
What are the two types of nucleic acids in a cell? are nucleic acids heteropolymers or homopolymers?
A
DNA (Deoxyribonucleic acid) and RNA (ribonucleic acid)
they are heteropolymers
3
Q
What are the major classes of biomolecules?. Which polymers combine to make up major class of biopolymers? What are the polymers made of?
A
biomolecules: Nucleic acids, proteins, carbohydrates, lipids.
Biopolymer: nucleic acids (nucleotides) , proteins (amino acids)and polysaccharides (sugar monomers)
4
Q
What is the longest polymers cell? Specifically which one?
A
Longest polymer is nucleic acids, specifically DNA (Deoxyribonucleic acid). They have large macromolecules with hundreds of millions of monomers.
5
Q
What are the main structures of nucleotides? How are nucleotides connected to another?
A
Phosphate group, 5-carbon sugar and nitrogen-containing base.
Nucelotides are connected to one another through phosphodiester bonds between 5’ and 3’ carbon atoms of sugar rings via phosphate group.
6
Q
What kind of polymers are RNA And DNA? How do DNA and RNA differ from each other?
A
Heteropolymers.
DNA have the same sugar and phosphate group, but bases have 4 different types.
DNA and RNA differ by sugar molecules and one base.
DNA- has deoxyribose sugar and bases: Adenine, thymine, guanine and cytosine
RNA- has ribose sugar (pentose) and bases: Adenine, URACIL, guanine and cytosine. (DNA and RNA share same 3 bases)
7
Q
What is a key element in nucleotide structure that connects all parts of nucleotide and is crucial for polymerization of nucleic acids? How are the carbons on this structure described?
A
Cyclic RIBOSE molecule.
on each numbered carbon on sugar of nucleotide, is followed by prime mark.
8
Q
How is the ribose attached to all other parts of nucelotide?
A
The base is attached to first carbon in ribose sugar, meanwhile the phosphate group is attached to 5th carbon in sugar.
9
Q
How do you differentiate between the sugars in RNA vs DNA? What conformation does each sugar have in common?
A
In RNA, there is an OH on BOTH the 2nd and 3rd carbon in ribose sugar.
IN DNA, there is ONLY OH on 3rd carbon and H on 2nd carbon.
Both DNA and RNA have sugar in Beta conformation (OH sticks above ring plane).
10
Q
What is the N-glycosidic bond? Which bases are pyrimidines and purines and where do each of them from N-glycosidic bonds?
A
Bond formed between base on first carbon on pentose sugar.
Pyrimidines- Cytosine, Uracil and Thymine
Purines- Adenine and Guanine (carbonyl)
Pyrimidines form N-glycosidic bond through 1st nitrogen atom, while purines use 9th atom.
11
Q
What’s the difference between thymine and uracil? which nucleic acid has thymine vs uracil?
A
Thymine- contains methyl group and carbonyls.
Uracil- has NO methyl group; also has carbonyls.
Thymine in DNA and uracil in RNA.
12
Q
What is the universal cellular energy intermediate and substrate for RNA?
A
ATP (Adenosine Triphosphate)
13
Q
How many phosphates can nucleotides attach to their sugars? What are the only phosphates cells use for making DNA or RNA (nucleic acid polymerization)?
A
Nucleotides can have one, two or three phosphates attached to sugars.
Ex: AMP- one phosphate; ADP- 2 phosphates
ATP- three phosphates
Cells only use TRIPHOSPHATES for making DNA and RNA
14
Q
Compare and contrast nucleoside vs nucleotide, and also explain how nucleotides are abbreviated.
A
Nucleoside- BASE and SUGAR
Nucleotide- BASE, SUGAR and PHOSPHATE
Also, nucleotides are abbreviated by three capital letters: AMP- adenosine monophosphate UDP= uridine diphosphate
15
Q
What are the substrates for DNA and RNA synthesis? where do the triphosphates carry their chemical energy?
A
substrates DNA- dATP, dGTP, dCTP,
Substrates for RNA- ATP, GTP, CTP, UTP
Triphosphates (like ATP) carry their chemical energy in hydrolyzed phosphoanhydride bonds.
16
Q
What part of nucleotide makes it negatively charged? Provide an example for this.
A
The phosphate group makes it negatively charged
DNA exists in form of negatively charged SALT RESIDUE (unconjugated base) associated with MAGNEUSIUM and POTASSIUM, other cations.
17
Q
Describe the 5’ end and 3’end and how they are used for making DNA or RNA.
A
5’ end- free triphosphate end of nucleic acid polymer.
3’ end- free, unbound hydroxyl group end (opposite to 5’ end) the 3’ end becomes hydrolyzed.
DNA or RNA strand starts with single triphosphate residue that is not hydrolyzed (5’ end) because next one is attached to 3’ hydroxyl.
18
Q
What are the similarities and differences for DNA and RNA.
A
DNA and RNA are both nucleic acids (seen in all cells) and both antiparallel.
DNA- DOUBLE STRANDED in both prokaryotes and eukaryotes (2 polymers attached to each other through H-bonds between nucleotide residues); right- handed double helix
RNA- SINGLE Stranded (some parts of RNA polymers may be double)
19
Q
What cellular structures may have double and single stranded DNA molecules and RNAs (ssRNA and dsRNA)?
A
Bacteriophages and viruses
20
Q
What is a reverse complementary sequence? List an example?
A
Reverse complementary sequence- sequence that is complementary to a particular sequence and written in 5’ to 3’ direction (left to right). Ex: what is the reverse complementary sequence for 5’ GCTTAGC 3’ ?
it is GCTAAGC
21
Q
How are nucleotide sequences always read (what direction)?
A
Always read from 5’ to 3’ direction
22
Q
How do complementary bases arrange DNA strands
What are the complementary base-pairing for DNA and RNA?
A
in anti-parallel orientation. one strand from 5’ to 3’ direction. Other strand from 3’ to 5’ direction.
DNA; A will be compl. to T; G to C ; T compl to A
RNA: A wiil be compl. to U; G to C; U compl to A.
23
Q
Describe the Watson Crick base pairing (how many bonds) and how it relates to nucleotides and rotation of double helix.
A
In DNA, watson-crick base pairs:
Adenine forms base pair with Thymine using 2 Hydrogen bonds.
Guanine forms base pair with Cytosine using 3 hydrogen bonds.
Linear order of nucleotide sequences in each DNA strand in double stranded DNA are complementary to each other (ex: 5’ GTCG 3’ is compl. to 3’ CAGC 5’
each base pair shifts relative to nearby base pair along axis (rotates) The rotation forms right handed double helix.
24
Q
Describe the kind of helix that double stranded DNA is and how many base pairs allow for one turn of double helix in DNA.
A
dsDNA is right handed helix
10 base pairs allow for one turn of double helix in DNA.
25
How are the grooves in the DNA double helix defined? Differentiate between the minor and major grooves of DNA Double helix.
The grooves are defined by spatial orientation of nucleoside monophosphate residue along helix.
Major groove- wider and provides access to BASES., for proteins that bind to SPECIFIC nucleotides sequences.
Minor groove- formed by phosphate, forming PHOSPHATE BACKBONE (neg. charged ridge on helix bind positive charge, allow for NON-SEQUENCE SPECIFIC RECOGNITION of DNA molecule by proteins like HISTONES.
many DNA and RNA recognizing proteins form both sequence-specific and non-sequence specific interactions with nucleic acids.
26
What is the purpose of cell division and what major process is involved? What is RNA? Where does it come from?
Purpose of cell division is to transfer genetic info encoded in DNA from parental cell to daughter cell from generation to generation.
This occurs through process of replication
RNA is a temporary copy of genetic information in DNA. It is synthesized in form of single stranded copy from one of DNA strands.
27
What is the genome and what form does it exist in?
| what is the role of proteins in the genome.
Genome is a collection of nucleotide sequences that encode protein-encoded RNAS (messenger RNAs (mRNAS), non-protein enconded RNAs (rRNAs, tRNAs, sRNAs, etc) and contain noncoding regions.
Genome exists in dsDNA (double-stranded DNA)
proteins encoded in genome take care of genome and synthesize all other biomolecules in cell.
28
Explain who Eduard Strausburger was and the things he observed under light microscope.
What did he find and draw out?
Eduard Strausburger was father or modern cytology, and in 1880s, documented a cell division that was observed for over 2 hours under light microscope.
He drew separation of chromatids (condensed chromosomes after duplication) in detail
29
Explain the discovery of chromatin and mitosis and how it led to knowledge of chromosomes.
What did he observe?
Who later named chromosomes?
Walther Flemming discovered chromatin and mitosis in 1878. He used ANILLINE dyes to find a structure that strongly absorbed basic dyes in the nucleus (basophillic substances), which he named chromatin .
He also observed that chromatin forms to thread-like structures in cell nucleus right before cell has to divide
German Wilhelm Von Waldeyer Hartz later named the thread like structures chromosomes.
30
Who is Theodore Bovery? What did he discover? What were his theories proposed?
Bovery discovered the CENTROSOME in 1888, and described it as special organ of cell division
Theories proposed:
-all chromosomes have to be present in order for proper embryonic development of sea urchin
-cancerous tumor cells begins with single cell where make up of its chromosomes are scrambled, causing cells to divide uncontrollably.
-proposed existence of cell cycle checkpoint, tumor suppressor genes, oncogenes, mitoses and uncontrolled growth may be caused by radiation, physical or chemical insults or microscopic pathogens.
31
What was main importance of Bovery's discovery and other scientists? What theory did scientists have in mind by 1920s?
All observations and discoveries made by Bovery and other scientists paved avenue for research directed towards further investigation of molecular bases of genes and inheritance
by 1920s, scientists convinced genes are located on the chromosomes (however, issue was Chromosomes are made of DNA and proteins).
32
Describe the three experiments that allowed for discovery of DNA as hereditary material and proof that genes are made of DNA. Be sure to includes scientists and dates.
1928- Experiment show hereditary material can be chemical substance- Frederick Griffith showed heat-inactivated bacteria that cannot infect anything can pass pathogenic factor to harmless bacteria (this experiment helped pave pathway to chemically isolate such substance).
1944- experiment showed DNA as hereditary material
Oswald Avery and others publish paper "Purification and Physical Characterization of Active Transforming principle, to offer proof that purified DNA extracted from pathogenic strain can act as genetic material (when isolated from one strain, DNA could transform its properties onto another strain)
1952- experiment using phages allowed for proof that genes are made of DNA. Martha chase and Alfred Hershey- analyzed viruses and labeled them with radioactive isotopes for DNA and Proteins. Noticed viruses grown in radioactive DNA contained radioactive DNA (not proteins)
together with studies by Avery, Macleod, McCarthy and others, evidence for case that DNA is agent of heredity and material carrier for genes.
33
What is Chargaff's Rule? Explain who Erwin Chargaff is other observations and theories he has?
Erwin Chargaff worked on nucleotide composition of DNA of various species like sea urchins.
Chargaff's rule- there is 1:1 ratio or relative amount of nucleic acids for amount of purines and pyrimidines. The amount of purines = amount of pyrimidines Also, relative content of A always equals content of T and content of G always equals the content of C.
He also suggested that guanine and thymine bases should predominantly exist in keto forms (favoring formation of H-bonds between A-T and G-C pairs equal in distance)
34
Explain who Rosalind Franklin was and her discoveries and theories, how they led to discovery of other scientists.
Rosalind Franklin worked on X-ray crystallography of DNA and obtained images of DNA Structure. Raymond Gosling also obtained images of DNA. She was able to discover DNA is right handed helix.
she also showed that Phosphate residues in DNA helix face outwards, while bases face inwards
Also observed that no molecules of water are inserted into DNA.
This observation led James Watson and Francis Crick to complete DNA Double helix model, as bases most likely form H-bonds between each other rather than with water.
35
describe how Watson and Crick were able to complete double helix model and how it has impacted study of genetics today.
Watson and Crick completed model in 1953, by fitting known information on structure and chemistry of nucleic acids into a model that allows formation of h-bonds between bases (base-pairing) with minimal tension between deoxyribose residues and overall chain. Showed DNA is a double helix
model provided mechanistic explanation of how genetic material replicated and preserved.
36
Describe Watson-Crick pairs in terms of their bonds and orientation.
Each base forms a set of H-bonds with a base on opposite strand based on rule of complementation.
Guanine interact with cytosine- 3 H bonds
Adenine interact with thymine - 2 H bonds.
Each Watson-Crick pair lays in plane that is parallel to plane that nearby base pairs form. Each pair also has rotational angle of 32 degrees (allow helix a complete turn over about 10 base pairs).
parallel orientation of base pairs allow pi orbitals from top and bottom of heterocycles to overlap along helical axis
37
What are Pi-stacking interactions? What are the three different types of pi-stacking interactions? How do they differ from Van Der Waals interactions?
non-covalent weak interactions that shared pi-orbitals of aromatic rings and other heterocycles form with each other.
three types: Sandwich, T-shaped and Parallel-displaced
They are not Van Der Waals interactions. Have different features:
-Pi clouds are capable of interacting with each other at large than (distance VDW permits)
-orientation of pi-clouds does not necessarily reflect adequate proximity of e- to nuclei (pivotal for VDW)
the exact nature of pi-stacking interaction is unknown, it is well-documented and provides DNA great stability and flexibility as well.
38
Describe the relationship between base pairing and DNA melting profile. How does temperature affect DNA? what is the characteristic melting point?
With an increase in temperature, DNA strands can be separated, and H-bonds will be disrupted.
Characteristic melting point is G-C dependent.
More GC pairs you have, more difficult it is to melt DNA, since it has 3 H-bonds.
However, A-T rich molecules, are easier to melt, since have only 2 H-bonds.
Sigmoidal melting curve shows effect of cooperativity. allows fragment with compl. sequences anneal to each other with high specificity.
39
What are two things used to detect DNA? Describe the process
DNA can be detected by UV absorption and Intercalating dyes.
DNA bases absorbs UV at 260 nm.
DNA loaded in agarose gel and DNA fragments can be separated by size in electrical field (gel electrophoresis)
stain with intercalating dyes like Ethidium Bromide helps visualize DNA (EtBr will intercalate into DNA)
Ethidium Bromide has very strong orange fluorescence (when exposed to UV) in complex with DNA. it will increase fluorescence by 20 fold when bound to DNA
40
Explain in detail, the semi-conservative mechanism of DNA replication, including its components. What is the process of DNA Replication?
DNA replication is semi-conservative, as newly formed double helix will have one OLD strand from parental helix and one NEW strand- hence semi-conservative.
DNA replication starts with separation of double helix into two single strands (S, S') that have fully COMPLEMENTARY base pairs (A to T on opposite strand, G compl to C)
each strand will serve as TEMPLATE for synthesis of daughter strand based on NUCLEOTIDE COMPLEMENTATION principle; forming finally new double helix (one old, one new)
41
What process must occur before each cell division? How does this process impact DNA in cell? Differentiate the mortality difference in bodies vs. germline cells.
DNA replication/synthesis must occur before any cell divides.
Mechanism of replication allows passage of faithful copy of genetic information during cell division.
Through many replication cycles, genetic information in double stranded DNA is passed from one cell to another in tissues in unicellular organisms, and through germ line cells from generation to generation in multicellular species.
Bodies (somas) are mortal. However, germline cells are not mortal (they are immortal) because information from these cells will be passed on from generations to generations.
42
Describe the three various models of DNA replication that were initally proposed.
1. Semi-conservative - had original double helix, where strands were separated, used as template to replicate and create another copy compl. to old strand. Have new helix with one old strand, one new strand.
2. Dispersive - each generation of replicated DNA molecules would have mosaic or mixture of both parental strands and newly synthesized DNA (half and half)
3. Conservative- parental strand remains intact after DNA replication, as one round of replication creates one original parent double helix and one entirely new double helix.
43
Describe the purpose, process and importance of Meselson-Stahl experiment, including scientists involved.
Meselson-Stahl experiment helped conclude that the correct mode of DNA replication was semi-conservative.
Matt Meselson and Frank Stahl had idea of using heavy nitrogen isotope to differentiate DNA species after round of replication through different density and thus flotation in cesium chloride density gradient
-First cell division cycle (transfer heavy to light medium) excluded Conservative model, but could not differentiate between semi-conservative and dispersive model (due to intermediate band (band halfway between heavy and light) appearing, could pertain to both models)
-Second round of replication (use heat to separate DNA strands), excluded the dispersive model because if dispersive model was correct, the second round of replication in light medium, would produce uniform band that would be slightly lighter than previous one. But the produced bands split on fully produced light bands and original band of intermediate weight.
44
Describe Meselson-Stahl's experimental process of proving that DNA replication is semi-conservative.
Process-
1. grow bacteria in one medium containing heavy N isotope, another containing light N isotope.
2. break open bacterial cells and load DNA into tubes containing salt cesium chloride
3. when these tubes are centrifuged, at high speed CsCl will form density gradient, DNA molecules float or sink in solution (distinguish between heavy and light isotopes based on position in solution)
4. Heavy DNA denser than light DNA and hence found at bottom of solution; while light DNA seen at top of solution.
5. Transfer bacteria grown in heavy medium to a light isotope medium. Observe what band occurs in solution after division for 1 hour.
6. Rule out proposed models based on results (ex: intermediate band in 1st replication rule out conservative model
45
what must occur to start DNA replication? Describe DNA molecules in prokaryotes and eukaryotes and how they will be separated. In which direction is DNA synthesized ?
DNA strands must be separated to start DNA replication.
DNA molecules are long in eukaryotes and circular in prokaryotes.
The DNA strands must be separated at specialized A-T rich areas called ORIGIN OF REPLICATION
Separation and synthesis of DNA at each replication origin continues in two directions through propagation of two Y-shaped Replication forks.
46
Differentiate between the speed of replication of chromosomes in eukaryotes and prokaryotes.
Prokaryotes- in particular, bacterial chromosomes are short and have ONE origin of replication
Eukaryotic chromosomes- have MANY origin of replication, and also multiple replication forks that allow for fast replication
separation/synthesis of DNA at each replication origin continues to spread until it reaches forthcoming rep. fork that was initiated at another, distant but nearby origin or in circular DNA, when forks move the half-circle and meet each other.
47
What helps open DNA double helix
Initiator proteins help open the double helix by recognizing nucleotide sequences and pulling DNA strands apart.
48
Explain how DNA replication is Bidirectional with use of replication forks. Which has faster replication fork movement, Bacteria or humans?
Once the two strands of DNA double helix are opened up, the two replication forks will move away from origin of rep. unzipping the DNA helix and copying DNA as they go.
Bacteria has faster replication fork movement as it is able to move at about 1000 nucleotides per second, while humans, fork moves 100 nucleotides per second.
49
What drives movement of replication forks?
DNA polymerase as it catalyzes addition of nucleotides to 3' end of growing DNA strand, using one of parental strands as template.
50
In which direction is a newly synthesized DNA strand in? explain mechanism behind copying of DNA strand. What bonds are formed?
New DNA strand copied in 5' to 3' direction. The base pairing principle of Watson and Crick enable DNA replication
Available 3' hydroxyl group at newly synthesized strand forms PHOSPHODIESTER bond with 5' phosphate group of incoming nucleotide. Ex: 3' end extends and growth occurs in 5' to 3' direction.
51
How does polymerization of nucleic acids happen? What are the substrates, products and bonds involved?
Polymerized through condensation of nucleoside triphosphates.
substrates for DNA (deoxyribonucleic acid) polymerization are DNA triphosphates.
product for Nucleic acid polymerization- nucleic acid strand and pyrophosphates
energy for reaction is derived from hydrolysis of high energy PHOSPHOANHYDRIDE bonds.
52
What enzyme catalyzes or carries out polymerization of nucleic acids?
DNA Polymerase (DNA DEPENDENT) catalyzes polymerization of nucleic acids. It uses DNA as a template to synthesize new DNA molecules (guides incoming DNA of triphosphate to pair with template strand). 5'end of triphosphate pair with 3' hydroxyl end of new strand.
53
Besides polymerizing nucleic acids, what other important functions do DNA polymerases have? How does it affect DNA Synthesis?
DNA polymerases can PROOFREAD DNA synthesis by stopping, BACKTRACKING, and excising (cutting) misincorporated nucleotides .
They are able to correct their own mistakes such as removing an incorrect nucleotide that was initially added to growing chain
proofreading -one of the major mechanism of FIDELITY of replication
Proofreading is why DNA synthesis always occurs in 5' to 3' direction (DNA polymerases only add nucleotide to 3' end
-
54
What would occur in a hypothetical 3' to 5' DNA Synthesis scenario, regarding proofreading?
in 3' to 5' DNA Synthesis scenario, proofreading will remove high-energy phosphates and block synthesis of DNA molecules.
removing incorrect nucleotide will block addition of correct nucleotide, since no high-energy phosphodiester bonds available at 5' growing chain to allow for polymerization to proceed.
Whereas, in 5' to 3' direction, even after polymerase proofreads and removes incorrect nucleotide, DNA strand can still continue to elongate.
DNA polymerase- self correcting enzyme.
55
Explain the orientation of DNA strands and how it relates to the replication fork. What direction does extension of new DNA strands go in? How does this affect replication fork?
DNA Strands are in ANTIPARALLEL orientation as they run in opposite directions. One template strand runs in 5' to 3' direction toward propagation of replication fork, while other strand is in 3' to 5' direction.
Extension of newly synthesized strands always occur in 5' to 3' direction.
Synthesis at replication fork is asymmetrical (one strand copied in direction of replication fork, right; and other strand extended in opposite.
56
Differentiate between the leading and lagging strands and be sure to explain what Okazaki fragments are and how they contribute to DNA Synthesis.
leading strand- strand at replication fork that can be extended continuously as it provides 3' end that can be extended in direction of propagating fork
Lagging strand- problematic; requires extension of DNA in backward direction that occurs intermittently, through forming short fragments of DNA.
Okazaki fragments- short fragments of extending DNA that are later ligated together once they are fully extended and cover template strand completely.
Both eukaryotic and prokaryotic cells have leading and lagging strands (b/c DNA polym, only synthesize 5' to 3' direction, proofread)
57
Differentiate between the role of RNA polymerase vs DNA polymerase and which are used in different scenarios.
DNA polymerase can only extend 3'end of an existing strand complementary to template strand.
However, RNA Polymerase, does not need base pairing end to replicate DNA at new strand. It can simply just join two ribonucleotide triphosphates together without base-pairing end.
A special RNA polymerase called PRIMASE can synthesize short RNA fragment (10 nucleotides long) called RNA PRIMER on template strand that is further extended by DNA Polymerase.
58
What is the function of primase?
Primase is an enzyme that can initiate DNA synthesis by synthesizing RNA by using DNA as a template. Primase is an example of RNA polymerase.
Like DNA polymerase, Primase synthesizes in 5' to 3' direction. However, primase does not need based paired 3' end as starting point.
59
How many primers are needed to synthesize DNA on leading strand compared to Lagging strand. Why?
Leading strand- may be synthesized from one single RNA Primer (need one primer to start replication at origin of replication)
Lagging strand- requires multiple primers (DNA synthesis on this strand is discontinuous, needs primers continuously to keep polymerizing).
60
Explain the process of DNA replication/synthesis on lagging strand, including the three enzymes involved. What must occur before the Okazaki fragments are joined together?
Process- (produce continuous new strand from separate pieces of nucleic acid)
1.Primase (RNA Polym) synthesizes RNA Primer (3’ to 5’)
2. DNA polymerase adds nucleotides to 3' end of of new RNA primer (Synthesize Okazaki fragment)
3. Nuclease removes RNA primer
4.Repair Polymerase (DNA polym) replaces RNA primer with DNA
5. DNA ligase seal nick and join Okazaki fragments together (join 5' phosphate end of one DNA fragment with 3' hydroxyl end).
Before joining the Okazaki fragments, the RNA Primer degrades, as RNA polymerase is capable of 5' to 3' exonucleolytic activity.
61
How does DNA ligase function in joining Okazaki fragments on lagging strand?
DNA ligase uses one ATP molecule to activate single phosphate at 5' end of Okazaki fragment. ATP is hydrolyzed, binding to 5' end and AMP is then released from cell, and eventually 5'end joins 3'end of another fragment.
62
Describe what a replisome is and its function, including all of the proteins that are part of the replisome.
A replisome is a complex protein machine that carries out DNA replication. Proteins work together at replication fork to synthesize new DNA
proteins composing replisome:
1. DNA Polymerase- catalyze addition of nucleotides to 3' end of growing strand of DNA using parental DNA strand as template
2. DNA helicase- use energy of ATP hydrolysis to unwind DNA double helix ahead of replication fork
3. Single-stranded DNA binding proteins- bind to single stranded DNA exposed by DNA helicase, prevent base pairs from reforming before lagging strand replicated
4. DNA topoisomerase- produce transient nicks in DNA backbone to relieve tension built up by unwinding of DNA ahead of DNA helicase.
5. Sliding clamp- keep DNA polymerase attached to template, allowing enzyme to move without falling off as it synthesized new DNA
6. Clamp loader- uses energy of ATP hydrolysis to lock sliding clamp onto DNA.
7. Primase- synthesize RNA primers along lagging strand template
8. DNA ligase - use energy of ATP hydrolysis to join okazaki fragments made on lagging strand template.
63
What is the function of Topoisomerase and how does it affect DNA Double helix? How does this enzyme apply to cancer cells?
Topoisomerase- resolves torsional stress during unwinding of Double helix (by helicase).
It creates a nick in DNA backbone to allow free rotation of DNA around single strands opposite to nicks.
Since there are higher than normal cell division rate and replication frequency in cancer cells, topoisomerase inhibitors are powerful chemotherapeutic agents
supercoiling also helps reduce torsional stress.
64
How many replication forks are present at each replication origin?
Two replication forks.
65
What enables DNA replication?
Base pairing(Watson-Crick) in Double stranded DNA
66
Describe the end problem of linear DNA, and also include the role of telomeres and telomerase in fixing this problem.
End problem of Linear DNA- The primer at very end of lagging strand cannot be substituted with DNA fragment synthesized by DNA polymerase.
Once the last primer is removed, the lagging strand will be incomplete (dna polym can only extend RNA primer, cannot start synthesis without it).
As a result, with each round of replication and cell division, DNA ends will get shorter and shorter.
An enzyme called Telomerase (conserved ribozyme) can fix this issue. Telomerase has RNA sequence with 5' TTAGGG 3' repeats that is complementary to DNA strand sequence 3' AATCCC 5'.
Telomerase can bind to and add telomere repeats to 3' end of template strand, allowing DNA Polymerase to lengthen newly synthesized lagging strand.
67
how many times is sequence of telomere repeats repeated in human chromosomes? What is the average telomere length from birth to old age?
In human chromosomes, sequence of telomere repeats can be repeated about 2,500 times
telomere length declines from 11,000 base pairs at birth to 4,000 base pairs at old age.
68
Define the role of a gene and what they encode, as well as mention what disrupts genes and products formed.
Genes are all of the DNA that encodes a primary sequence of some final gene product.
Most genes encode for proteins. some genes encode for special types of RNA that DO NOT encode proteins.
Genes are interrupted by non-coding sequences
Genes can produce more than one gene product by a number of mechanisms.
69
What are chromosomes? What is a nucleotide sequence of DNA composed of? Explain what comprises of E.coli genome.
Chromosomes are long stretches of DNA that contain genes
nucleotide sequence of DNA has gene sequences and intergenic non-coding regions.
E.coli genome is single circular DNA (Chromosome) of size of 5.4 million base pairs and 5,416 genes.
70
Describe the human genome in terms of size and compositions. What is size of human chromosome? What is size of human genome?
Eukaryotic have multiple linear chromosomes (where genes are located).
30% of mammalian and human genome encode for genes (45% transposons, 25% other)
The average size of human chromosome is about 130 million base pairs (2 inch long).
Size of human genome is 3 billion base pairs for 23 unpaired chromosomes.
71
Describe the appearance of chromosomes during mitosis and also define what a centromere and what other structures are connected to it. How are chromosomes separated and how do arms come in to play?
During mitosis, chromosomes are condensed and highly visible. In interphase, cell grows, replicates its DNA and prepares for mitosis.
right before cell division (during mitosis), chromosomes become condensed for easy separation.
centromere- non-coding region in middle of chromosome that is used as attachment site for microtubules to separate sister chromatids from each other to daughter cells.
Sister chromatids- condensed chromosomes after duplication.
centromere region separates chromosomes on arms
The arms of chromosome are usually asymmetric (different in size). smaller arm is called p (petite) and larger arm is called q (q follows p)
72
How are chromosomes numbered?
They are numbered by length
73
What is a karyotype? Explain how chromosome changes can lead to clinical diseases, and provide examples.
Karyotype-collection of condensed, mitotic chromosomes with distinct appearance that is specific among species.
difference in chromosome morphology (extension of arms, duplication) can point on genetic abnormalities.
Ex: Down syndrome (chromosome 21 trisomy), ataxia- extension of chromosome 12; inherited leukemia- Philadelphia chromosome (rearrangement between chromosome 9 and chromosome 22) .
74
Explain the role of chromosomes during evolution and how it relates to complexity of organisms. Provide examples.
During evolution, chromosomes are highly mobile and undergo constant rearrangement.
similar to genome size, the length of chromosomes and the number does not always correlate with complexity of an organism.
Ex: Chinese Muntjac and Indian Mutjac, are both little deers with similar appearances that have different karyotypes.
Chinese munt- has many, short chromosomes, while Indian munt- has few, long chromosomes. Despite different karyotypes, the two deer are very similar.
75
Describe the appearance and structure of chromosomes during interphase? What contributes to their structures?
During interphase, chromosomes are not randomly distributed in the nucleus.
They organized by nuclear lamins, which are intermediate filaments (or fine woven meshwork of special cytoskeletal proteins) that form lining on inner surface of nuclear membrane; providing structural and functional support for chromosomes, and organizing them.
76
How many chromosomes contribute to formation of nucleolus and how do they contribute?
10 chromosomes contribute to formation of nucleolus.
| Each of 10 chromosomes contribute a loop containing rRNA genes to nucleolus.
77
Explain the different condensation levels of DNA during interphase, and be sure to differentiate between heterochromatin and euchromatin.. what is the benefit of changes in chromosome structure? Which form of chromatin is not active ?
condensation level of DNA within chromosome varies,
Interphase chromosomes contain both condensed form (Heterochromatin)and more extended forms of chromatin (euchromatin).
Changes in chromosome structure allows access to DNA.
Heterochromatin - is normally suppressed, not active and is associated with nuclear lamina
78
Differentiate between the structure of chromosomes in interphase vs mitotic phase. What does loose chromatin resemble structurally?
While mitotic chromosomes are highly condensed, interphase chromatin is more loose.
EM and biological analysis of chromatin shows that even the most loose chromatin is tightly associated with proteins, forming a structure similar to beads on a string.
79
What are the basic units of eukaryotic chromosome structure? Explain what it is and how what its composed of and also
Nucleosome
A nucleosome is a nucleoprotein structure with four homodimers of histones (speciailzed Arginine and Lysine rich proteins that bind DNA nonspecifically) that form oligomeric complex around which thread of DNA wraps (making 1.7 turns )
80
What are nucleosome core particles composed of and what causes them to be released? How many core histone proteins are there what are they?
Nucleosome core particles composed of 147 bp (base pairs) that wrap around histone octamer.
The nucleosome core are protected by nucleases that digest DNA (DNAse).
Adding DNAse to bead-on string chromatin will digest linker DNA that is not protected by histones and thus releases core particles.
there are total of 4 main core histone proteins. The main histones are: H2A, H2B, H3 and H4.
81
Describe the process of forming nucleosomes and all of histones involved.
Formation of nucleosome is spontaneous process where histones bind cooperatively.
Besides the 4 core histones (H2A, H2B, H3 and H4), there are other MINOR histones that are histone-like and associated proteins that define chromatin packaging.
82
Which histone brings nucleosomes together to form 30 nm fiber?
Histone H1. H1 will constrain additional 20 bp (base pairs) of DNA to bring nucleosome together.
The compaction allow packaging of nucleosomes in 30 nm fibers. (level of chromatin compaction may vary , regulated by many mechanisms).
83
How is euchromatin structured and what other parts are involved?
Euchromatin is structured by loop domains with matching specific DNA sequences and chromosome-loop forming clamp proteins?
84
Describe the different levels of DNA packaging in chromosomes.
DNA packing occurs on several levels in chromosomes.
first have short region of DNA double helix, then turns to "beads on a string" form of chromatin.
The chromatin fiber is then formed with packed nucleosomes. Chromatin fiber then folded into loops.
Each DNA molecule has been packaged into mitotic chromosome that is 10,000 fold shorter than its fully extended length.
85
What role do Chromatin-remodeling complexes play in DNA? How does this affect nucleosomes and condensation?
Chromatin-remodeling complexes locally reposition DNA on nucleosome, using energy of ATP hydrolysis.
Complex slides DNA along histone octamers (like thread slid along spool.
As a result, nucleosomes end up being spread farther apart from each other due by increasing size of linker DNA.
Hence de-condensation achieved without dissociation of histones from DNA.
86
What other forms of modification do Histones partake in chromatin structure.
Histones partake in acetylation, and also multiple covalent modifications like controlling gene expression, gene silencing, heterochromatin formation.
Ex: Histone H3 modification (adding 3 methyl groups leads to heterochromatin formation and gene silencing.
87
What is position effect? When does it often occur? How can chromatin-remodeling complexes silence genes?
Position effect on chromatin due to close proximity of active, euchromatin genes to heterochromatin regions
This may lead to condensation of euchromatin and thus SILENCING of genes.
This often occurs when there are chromosomal rearrangements (swapping or inversion) of the parts, and the area that used to be pure euchromatin (not condensed), ends up next to heterochromatin and inactivated.
88
Explain how the position effect occurs in Drosophila X chromosome.
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The White (w+) gene defines the normal, wild-type color of drosophila eye, which is RED
This white (w+) gene is normally located in euchromatin (uncondensed) region of drosophila X chromosome far away from heterochromatin region of CENTROMERE
But chromosomal inversion may result in w+ gene being located adjacent to heterochromatin regions that may lead to condensation of euchromatin and thus SILENCING of genes.
when w+ gene is silenced in some cells, there will be no pigment. This occurs whenever chromosomal rearrangement (inversion, swap ) happens, and euchromatin ends up next to heterochromatin, and inactivates.
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89
How are eukaryotic cells capable of silencing whole chromosome? Who does this especially pertain to and how are they described as because of this?
During early embryonic development of FEMALES, in embryonic cells, one of two X chromosomes will get condensed to heterochromatin and will thus be inactivated.
Process is spontaneous as cell will decide randomly which X chromosome, maternal or paternal gets inactivated. But all cells and tissues that originated from that cell will continue to have same X chromosome inactivated.
Hence all mammalian females are MOSAIC as their organs and tissues express either paternal or maternal X chromosome associated genes.
90
How are bacterial genomes structured? what stabilizes them?
Bacterial chromosomes are not isolated by membrane and not bound to histones.
bacterial genomes are structured through set of supercoils that span about 10,000 base pairs. They are stabilized by specialized proteins called NUCLEOIDS (nucleus-like arrangement) that anchors DNA to plasma membrane from the inside and assists in DNA REPLICATION.
91
Explain how mitochondrial genomes differ in structure from bacterial genomes and the unique protein it is composed of.
Like bacterial genomes, mitochondrial genomes are heavily structured in from of nucleoid
Difference:
mitochondrial genome- small circular, and arranged as compact, protein-associated and membrane anchored nucleoid in matrix (cryo-electron microscopy)
A special protein called TFAM- covers almost entire mitochondrial DNA , forming multiple U turns on DNA, creating sufficient level of compaction.
TFAM (mitochondrial transcription factor A)
92
What kind of structures are eukaryotic DNA packaged into?
multiple linear chromosomes
93
What are the functions of chromosomes?
to organize and carry genetic information (genes)
94
Specialized DNA sequences are required for what two processes?
DNA replication and Chromosome segregation
95
What are the basic units of eukaryotic chromosome structure?
NUCLEOSOMES.
